Construction And Identification Of Mycoplasma Bovis Strain 08M Mutant Library

Mycoplasma bovis(M.bovis)is a pathogen that can cause a variety of diseases in beef cattle,dairy cows and calves.Clinical symptomsinclude pneumonia,mastitis,arthritis,meningitis,keratoconjunctivitis and genital disorders.M.bovis infection is widespread all over the world,which has caused huge economic loss to dairy products and beef products industry.The lack of genetic manipulation of M.bovis has been hindering the study of its pathogenic mechanism.Transposon is an effective tool for genetic manipulation of M.bovis at present.Transfer the transposon into M.bovis cells by electroporation can result in random insertion and inactivation of genes,bringing light to the study of gene function and pathogenesis of M.bovis.The purpose of this study was to construct a mutant library of Mycoplasma bovis(M.bovis)strain08M by transposon insertion,which could provide a platform for identification of the virulent genes of the pathogen.First,the color change units(CCU)and the gentamicin(Gm)minimum inhibitory concentration(MIC)of M.bovis strain 08M were determined,and then the plasmid pISM2062 carrying transposon Tn4001mod was introduced into the cells of strain 08M by electroporation.Finally,the mutant library of strain 08M was constructed by mutant clones screening,culturing,and PCR identification.Ten clones were selected and passaged 10 times to detect the stability of transposon by PCR,and two clones were selected to detect whether the transposon were inserted with second or multiple times by Southern-blot.A total of 28 pairs of primers related to the predicated virulence-related genes of M.bovis were designed and synthesized.A preliminary screening of the mutant library was performed using the Haystack screening method to screen the mutants of the gene of interest.The results showed that the initial concentration of strain 08M was 1.13×10~7 CCU/mL,and the gentamicin MIC for strain 08M was 16 ng/?L.A total of 445 colonies were picked up after electroporation.Of 380 grown colonies,263 were identified as positive Tn insertion clones by PCR.The positive rate of the mutant library was 69.21%(263/380).PCR identification results showed that the inserted transposon was stable.The results of Southern-blot showed that the transposon was not repeatedly inserted during the passage.No mutant strains of interested genes were screened from the mutant library using the Haystack screening method.In this study,the mutant library of M.bovis was constructed.The transposon is stably existed in the mutant strains.No mutant strains of interested genes were screened from the mutant library using the Haystack screening method.